The primary areas of investigation for the Laboratory of Hepatitis Research are targeted to the hepatitis C virus (HCV). The HCV effort has been directed at understanding the immunobiology and pathogenesis of HCV and strategies for vaccine development. As part of the vaccine and pathogenesis projects, we are developing a chimeric mouse/chimpanzee liver model that will be useful for a variety of studies that include HCV infection. The innate immune system, particularly the role of interferon and interferon resistance in HCV is also being studied. In order to be prepared for the possibility of SARS virus vaccine applications, a project on the biology of SARS virus has been initiated. Immunopathogenesis of HCV: Both CD4+ and CD8+ memory T cells are important in providing protective immunity from HCV reinfection. CD4+ T cells with a distinct cytokine profile contribute to HCV resolution in acute infections of chimpanzees. A subset of these cells that were CD4+ and CD8+ were specific for HCV,expressed IFN-gamma and IL-4. These highly activated but not cytopathic cells were important in the clearance of HCV in acute infections. Reinfection of resolved animals revealed that while the animals were not protected from reinfection, vigorous T cell responses could rapidly eliminate the second infection. If the responses were slower or weaker, the second infection could persist at low levels for extended periods of time, and if the T cells declined below detectability, the virus might reappear. HCV Vaccine studies: We have shown that antibody responses to surface antigens on HCV can control HCV viremia, but if antibody levels decline, the virus can reemerge and even become chronic. Anti E1/E2 antibody induced by vaccination, added to the T cell responses already existent in recovered chimpanzees was able to protect the animal from HCV induce liver disease as well as infection except for a very brief, very low level viremia. A vaccine designed to induce only T cell immunity to non-structural region antigens was shown to boost T cell responses in recovered chimpanzees. This resulted in a major attenuation of infection upon rechallenge relative to the infections seen in non-vaccinated rechallenged chimpanzees. This T cell vaccine was also able to significantly attenuate HCV infection and disease in a naive chimp. This vaccine was also tested as an immunotherapeutic in a chronically infected chimpanzee. Methods development: Chimeric mouse/chimpanzee liver models have been developed to study HCV virus infection. The original transgenic mice, ALB-uPA, which express the urokinase plasminogen activator gene under control of the albumin promoter crossed with SCID-beige immunodeficient mice can support human liver implants. However, these mice are very poor breeders, and homozygous ALB-uPA mice rarely survive long enough to perform useful experiments. We have developed a transgenic mouse that expresses the E. coli nitroreductase gene under the albumin promoter. These mice are healthy until given a nontoxic prodrug, CB1954, which is converted to a toxin by nitroreductase. These mice have now been bred to SCID-bg mice and once a colony is established, they can be tested for their ability to sustain chimpanzee or human liver to replace their own liver. Once established, the chimp liver can serve as a substrate for HCV replication. True antibody neutralization could be studied in these mice as well as a number of other aspects of HCV immunology and replication. Mechanisms of interferon (IFN) resistance in HCV: A new antiviral pathway has been discovered that may be important in the biology of HCV infections. ADAR, adenosine deaminase that acts on RNA, is induced by IFN. The subgenomic HCV replicons made from the relatively IFN resistant HCV genotyp 1b is exquisitely sensitive to IFN in vitro. In replicons containing cells treated with IFN, we have identified adenosine to inosine conversion (a function of ADAR) This RNA editing results in the rapid destruction of the inosine containing RNA. We have found that inhibitors of ADAR reduce the conversion of adenosine and protect the loss of replicon RNA in IFN treated cells. This newly described antiviral pathway have implications for therapeutic strategies. Natural history of HCV: Plasma samples from chimpanzees inoculated with as long as 28 years previously continues to be studied. The molecular evolution of HCV in chronically infected chimpanzees has identified several newly emerging mutations within the HCV RNA dependent RNA polymerase. Some of these mutations have been shown to enhance the activity of the polymerase in vitro, but others have nearly eliminated in vitro activity. These mutations are being studied in detail along with the the effect of other mutations occurring simultaneously in other genes coding for some of the non-polymerase proteins of the replication complex. Studies on the SARS virus: In collaboration with Kanta Subbarao and Brian Murphy of NIAID, we are looking at different methods for inactivation of the SARS virus that may be useful for development of vaccines, diagnostics as well as for protecting the blood supply. We also will establish in vitro neutralization assays to aid the evaluation of vaccine candidates and will study the potential for immune dependent enhancement of the clinical spectrum of SARS which may have a bearing on vaccine approaches. This project incorporates FY2002 projects 1Z01BK004001-10, 1Z01BK004002-10, and 1Z01BK004005-05.